Methods and apparatus for perfusion and environment control of microplate labware

ABSTRACT

Systems, methods, and apparatuses of controlling fluid flow are disclosed. An apparatus includes a first microplate having a first open portion and defining one or more first wells therein, a second microplate having a second open portion and defining one or more second wells therein, and a pneumatic lid constructed of styrene ethylene butylene styrene (SEBS). The pneumatic lid extends over the first open portion and the second open portion and includes one or more microfluidic channels that fluidly couple the one or more first wells to the one or more second wells. The pneumatic lid provides an airtight seal over the first microplate and the second microplate.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of International Application No.PCT/US2018/014447, filed on Jan. 19, 2018, which claims priority to U.S.Provisional Application No. 62/447,991, filed Jan. 19, 2017, both ofwhich are incorporated by reference herein in their entirety.

TECHNICAL FIELD

The present specification generally relates to enhanced in vitro cellculture, and more particularly, to systems, apparatuses, and methods forproviding integrated perfusion and atmospheric control of microplatelabware.

BACKGROUND

Currently, many in vitro cell culture techniques exist to provide amethod to keep biological cells alive ex vivo over extended timeperiods. For example, certain techniques include a static culture,manual batch feed in which cells are seeded on a cell culture vesselsuspended in media and placed in a temperature- and CO₂-controlledincubator. However, such techniques are not ideal for mimicking a truein vivo physiological microenvironment. For example, in a mammalianbody, the cellular microenvironment varies considerably from theconditions that can be stimulated in vivo. Therefore, because cells tendto be a product of their microenvironment, the in vivo cultured cellsare not a true representative of cells that occur in a physiologicalenvironment.

Some solutions to this issue require specialized labware, which isexpensive, not commercially available, and/or is particularly suitedonly for certain applications. Such solutions cannot utilize standardmicroplate labware, which is widely available for a multitude oflaboratory applications. In addition, other solutions are not accessibleto light microscopy, contain a limited throughput, contain a limitednumber of cell wells/chambers (e.g. <12 per microplate footprint), aredifficult to handle and/or load cells, lack atmospheric control, lack anability to control a flow rate, have transient flow rates, have alimited flow duration, have a requirement for re-circulation, have arequirement for mechanical tilting of the plate to extend the duration,and/or do not have independent well control (i.e., all wells undergoidentical perfusion treatment).

Accordingly, there exists a continuing need for an in vitro cell culturetechnique that allows for enhanced control of the cellularmicroenvironment using standard microplate labware, as well as systems,apparatuses, and the like for carrying out the technique while alsobeing able to integrate with standard microplate labware.

SUMMARY

In an embodiment, a pneumatic lid includes a body having one or moremicrofluidic channels. At least a portion of the body is constructed ofstyrene ethylene butylene styrene (SEBS). The pneumatic lid furtherincludes one or more first extension pieces fluidly coupled to the oneor more microfluidic channels and extending from the body and one ormore second extension pieces fluidly coupled to the one or moremicrofluidic channels and extending from the body.

In another embodiment, a pneumatic lid includes a first portion havingone or more first microfluidic channels that are configured to befluidly coupled to one or more first wells, a second portion having oneor more second microfluidic channels that are configured to be fluidlycoupled to one or more second wells that are separate from the one ormore first wells, and a removable bridge portion extending between thefirst portion and the second portion. The removable bridge portion, whencoupled to the first portion and the second portion, fluidly couples theone or more first microfluidic channels to the one or more secondmicrofluidic channels. The first portion and the second portion, whencoupled to the one or more first wells and the one or more second wells,respectively provide an airtight seal over the one or more first wellsand the one or more second wells.

In yet another embodiment, an apparatus includes a first microplatehaving a first open portion and defining one or more first wellstherein, a second microplate having a second open portion and definingone or more second wells therein, and a pneumatic lid constructed ofstyrene ethylene butylene styrene (SEBS). The pneumatic lid extends overthe first open portion and the second open portion and includes one ormore microfluidic channels that fluidly couple the one or more firstwells to the one or more second wells. The pneumatic lid provides anairtight seal over the first microplate and the second microplate.

In yet another embodiment, an apparatus includes a first microplatehaving a first open portion and defining one or more first wellstherein, a second microplate having a second open portion and definingone or more second wells therein, and a pneumatic lid extending over thefirst open portion and the second open portion. The pneumatic lidincludes one or more microfluidic channels that fluidly couple the oneor more first wells to the one or more second wells. The pneumatic lidprovides an airtight seal over the first microplate and the secondmicroplate.

In yet another embodiment, an apparatus includes a first microplatehaving a first open portion and defining one or more first wellstherein, a second microplate having a second open portion and definingone or more second wells therein, and a pneumatic lid. The pneumatic lidincludes a first portion extending over the first open portion, thefirst portion having one or more first microfluidic channels that arefluidly coupled to the one or more first wells. The pneumatic lidfurther includes a second portion extending over the second openportion, the second portion having one or more second microfluidicchannels that are fluidly coupled to the one or more second wells. Thepneumatic lid also includes a removable bridge portion extending betweenthe first portion and the second portion. The removable bridge portion,when coupled to the first portion and the second portion, fluidlycouples the one or more first microfluidic channels to the one or moresecond microfluidic channels. The pneumatic lid provides an airtightseal over the first microplate and the second microplate.

In yet another embodiment, a method of constructing an apparatus fortransferring fluid includes providing a first microplate having a firstopen portion and defining one or more first wells therein, providing asecond microplate having a second open portion and defining one or moresecond wells therein, and placing a pneumatic lid constructed of styreneethylene butylene styrene (SEBS) over the first open portion and thesecond open portion such that one or more microfluidic channels withinthe pneumatic lid are fluidly coupled to the one or more first wells andthe one or more second wells. The pneumatic lid provides an airtightseal over the first microplate and the second microplate.

In yet another embodiment, a method of constructing an apparatus fortransferring fluid includes providing a first microplate having a firstopen portion and defining one or more first wells therein, providing asecond microplate having a second open portion and defining one or moresecond wells therein, placing a first portion of a pneumatic lid overthe first open portion such that one or more first microfluidic channelswithin the first portion are fluidly coupled to the one or more firstwells, placing a second portion of a pneumatic lid over the second openportion such that one or more second microfluidic channels within thesecond portion are fluidly coupled to the one or more second wells, andplacing a removable bridge portion between the first portion and thesecond portion of the pneumatic lid to fluidly couple the one or morefirst microfluidic channels to the one or more second microfluidicchannels.

In yet another embodiment, a system for transferring fluid includes afirst microplate having a first open portion and defining one or morefirst wells therein, a second microplate that is separate front thefirst microplate the second microplate having a second open portion anddefining one or more second wells therein, a pneumatic lid, and one ormore valves. The pneumatic lid is constructed of styrene ethylenebutylene styrene (SEBS) which forms a reversible and gas impermeablebond with the first microplate and the second microplate. The pneumaticlid includes a first portion extending over the first open portion, thefirst portion including one or more first extension pieces extendinginto the one or more first wells of the first microplate and one or morefirst microfluidic channels that are fluidly coupled to the one or morefirst wells via the one or more first extension pieces, a second portionextending over the second open portion, the second portion including oneor more second extension pieces extending into the one or more secondwells of the second microplate and one or more second microfluidicchannels that are fluidly coupled to the one or more second wells viathe one or more second microfluidic channels, and a removable bridgeportion extending between the first portion and the second portion. Theremovable bridge portion, when coupled to the first portion and thesecond portion, fluidly couples the one or more first microfluidicchannels to the one or more second microfluidic channels. The one ormore valves are fluidly coupled to the pneumatic lid and configured toselectively control fluid flow within the one or more first microfluidicchannels and the one or more second microfluidic channels. The fluid istransferred between the first microplate and the second microplate viathe pneumatic lid and the one or more valves.

These and additional features provided by the embodiments describedherein will be more fully understood in view of the following detaileddescription, in conjunction with the drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The embodiments set forth in the drawings are illustrative and exemplaryin nature and not intended to limit the subject matter defined by theclaims. The following detailed description of the illustrativeembodiments can be understood when read in conjunction with thefollowing drawings, wherein like structure is indicated with likereference numerals and in which:

FIG. 1A depicts an exploded schematic cross-sectional view of anillustrative apparatus for providing integrated perfusion andatmospheric control of microplate labware according to one or moreembodiments shown and described herein;

FIG. 1B depicts a schematic cross-sectional view of an illustrativeapparatus for providing integrated perfusion and atmospheric control ofmicroplate labware when coupled to the microplate labware according toone or more embodiments shown and described herein;

FIG. 1C depicts an exploded schematic cross-sectional view of anillustrative apparatus having a bridge portion, the apparatus providingintegrated perfusion and atmospheric control of separate microplatelabware, according to one or more embodiments shown and describedherein;

FIG. 1D depicts a schematic cross-sectional view of an illustrativeapparatus having a bridge portion when coupled to separate microplatelabware according to one or more embodiments shown and described herein;

FIG. 2 depicts a block diagram of illustrative hardware that may be usedto control an apparatus for providing perfusion and atmospheric controlof microplate labware according to one or more embodiments shown anddescribed herein;

FIG. 3A depicts a schematic view of an illustrative pneumatic lid thatincorporates solid polymer plugs at a destination plate interfaceaccording to one or more embodiments shown and described herein;

FIG. 3B depicts a detailed view of the solid polymer plugs of FIG. 3A atthe destination plate interface according to one or more embodimentsshown and described herein;

FIG. 4A depicts a schematic view of an illustrative pneumatic lid thatincorporates polymer tubes at a destination plate interface according toone, or more embodiments shown and described herein;

FIG. 4B depicts a detailed view of the polymer tubes of FIG. 4A at thedestination plate interface according to one or more embodiments shownand described herein;

FIG. 5A depicts a schematic view of an illustrative pneumatic lidinterface well mapping configuration according to one or moreembodiments shown and described herein;

FIG. 5B depicts a schematic view of another illustrative pneumatic lidinterface well mapping configuration according to one or moreembodiments shown and described herein;

FIG. 5C depicts a schematic view of yet another illustrative pneumaticlid interface well mapping configuration according to one or moreembodiments shown and described herein;

FIG. 6A depicts a schematic view of an illustrative apparatus for wastecollection according to one or more embodiments shown and describedherein; and

FIG. 6B depicts a schematic view of an illustrative pneumatic lidinterface well mapping interface for waste collection according to oneor more embodiments shown and described herein.

DETAILED DESCRIPTION

Referring generally to the figures, embodiments described herein aredirected to in vitro cell culture techniques that utilize methods,systems, and apparatuses to provide control of a cellularmicroenvironment using standard microplate labware by providing a devicewith a large well throughput, atmospheric control, active fluidperfusion of any and all wells, extended experimental perfusionduration, and compatibility with simultaneous microscopic imaging. Theembodiments described herein generally include a plurality ofmicroplates that are fluidly joined together via a pneumatic lid that isat least partially constructed of a thermoplastic elastomer that forms areversible and gas impermeable bond with the plurality of microplates.More particularly, the thermoplastic elastomer is or includes styreneethylene butylene styrene (SEBS).

As used herein, the terms “microplate labware” or “standard microplatelabware” refer to labware that is generally understood and used for thepurposes of cell culture, particularly mammalian cell culture.Illustrative examples of such labware include, but are not limited to,open culture labware vessels such as microplates, T-flasks, petridishes, or the like, particularly vessels that are suited for batch-feedprocesses.

The various techniques of the present disclosure may include certain invitro cell culture techniques that keep biological cells alive ex vivoover extended time periods. Illustrative techniques may include, forexample, cell culture vessels generally fabricated from injection moldedplastic including multi-well plates, T-flasks, petri dishes, or thelike. Such cell culture vessels may be configured to breathe, i.e., theinternal gas concentrations are intended to equilibrate to the gaseousenvironment in which they are placed. Certain design features describedherein may filter this gas exchange in an attempt to eliminate air-bornecontaminants such as bacteria or fungal spores. Specialized media,supplements, and saline solutions customized to the biology under studymay be used with a prime objective of providing the essential basicsalts, amino acids, nutrients, and growth factors for the purpose ofkeeping cells healthy. In addition, the methods, systems, andapparatuses described herein may include maintenance of physiologicaltemperatures (e.g., about 37° C.), maintenance of physiological pH byusing, for example, bicarbonate buffered solutions and a specificpartial pressure of carbon dioxide (e.g., about 5%), and/or maintenancein a humidified environment, such as, for example, an environment havinga relative humidity of about 90 to about 95%. Such a humidifiedenvironment may be necessary to reduce evaporation of fluid in the labculture vessels. Such evaporation could potentially result in adversechanges in osmolarity of the solution, which may cause harmful effectsto the cells.

Certain cell feeding techniques, such as a technique that involves astatic culture, manual batch feed, cells may be seeded on a cell culturevessel suspended in media and placed into a temperature controlledincubator for temperature and CO₂ maintenance, as described herein.Adherent cells may fall to the bottom of the vessel and may subsequentlyattach to the cell surface. Non-adherent cell types may be cultured inthis manner, or may be cultured in Spinner-Flasks that maintain thecells in suspension via constant stirring. To minimize humanintervention, the media may carry an excess of nutrient constituentssuch that the cells remain viable for a particular time period, such as,for example, at least about 24 hours from the initial feeding. Duringthis time, nutrients may be consumed by the cells and waste products maybe generated. Once the nutrients have been consumed and/or the wasteproducts have built up to levels which can affect the cells healthdirectly, a user may remove the vessel from the incubator and replaceall or a portion of the cell media, use the cells in an experiment, orharvest the cells for further uses or seeding of new vessels. It shouldbe understood that such cellular manipulation (feeding, harvesting, orpassaging) may be performed within a sterile biological cabinet attypical atmospheric conditions and at room temperature.

Such cell feeding techniques may not mimic the in vivo physiologicalmicroenvironment. This is because, in vivo, tissues obtain a steadystate supply of nutrients as fed by the arterial system (source) anddrained by the venous and lymphatic systems (sinks). The actualinterstitial flows (tissue flow) which feed cells are tissue dependentand based on the local metabolic demands of the tissue. This tissuedependence is accommodated by the arterial spacing (higher metabolicdemand tissues have closer capillary spacing), pressure differentialsdominated by local hydrostatic and osmotic pressure differences betweenthe capillaries/venuoles and the tissue, and the fluid permeability ofthe local tissue environment. Interstitial flows are typically quitesmall, on the order of tens of microns per minute, and as such, the flowof nutrients and waste removal is a slow, steady-state concentration ofnutrients and waste products.

Use of such cell feeding techniques may result in cells that are aproduct of their cultured microenvironment, and vice-versa. For example,cell metabolism of cells in a cultured microenvironment may differ fromcells that occur naturally in vivo. Cellular catabolic metabolism canshift from glycolysis (glucose input) to oxidative phosphorylation(pyruvate and oxygen) to glutaminolysis (glutamine input) depending onthe availability of such nutrients. In addition, organisms can adapt totransient supply/demand variations by storing away fuel via glycogenstorage, fatty acid anabolism or the Pentose Phosphate Pathway or callupon fuel stores via lipolysis and fatty acid catabolism. Typical wasteproducts such as lacate may become sources of fuel under certainconditions. In contrast, cell culture media may supply about 3 to about10 times excess concentrations of these fuel sources in order to keepcells viable for multiple days. In addition, cell culture media maycontain excess levels of amino acids and vitamins in much the samemanner. Such cell culture media may also be optimized such that the cellculture media is broadly applicable to many cell types. Moreover,batch-feed processes may be optimized for convenience, such as, forexample, requiring manually feeding only every few days.

Similarly, the cultured cells can have an effect on the localmicroenvironment. Cells may secrete waste products, growth factors,cytokines, and other signaling molecules. Some secreted products mayhave an impact on the cells that secreted the products or on other cellsvia autocrine or paracrine signaling. If such secreted products areallowed to build up within a static, batch feed feeding process,concentration gradients and transients form, which may not berepresentative of the in vivo condition in which a steady flow removeswaste products in a more stable, homeostatic condition.

In addition, some tissues in the body have oxygen concentrations whichare less than atmosphere concentrations (e.g., about 21%). For example,typical concentrations in the liver are about 3% to about 9% and in thebrain are about 2% to about 7%, and an actual concentration may form adecreasing concentration gradient in tissues that are located fartheraway from a supply capillary. Since oxygen is a necessary input tomammalian cell metabolism via the citric acid cycle or the Krebs cycle,changes in an available oxygen concentration may have an effect on cellphenotype.

Various cell culture techniques may require a manual intervention tofeed cells, which may be time consuming and may contaminate the cellsand/or the media. Microplates may be configured to breathe by exchangingair around the perimeter of the microplate. However this may create anon-uniform air flow and may cause greater evaporation around the edgewells of the microplate, relative to the center wells of the plate. Sucha non-uniform evaporation may cause osmolarity differences and edgeeffects on microplate cell cultures.

Some cell culture devices may incorporate integrated perfusion, such asmicrofluidic cell culture devices that incorporate microfluidic cellchambers with integrated channels and valving. Illustrative microfluidicfeatures generally include design elements having feature dimensions onthe order of tens to hundreds of microns. However, microfluidic deviceshave not been commercially viable because such devices are muchdifferent to work with than standard microplate, or “open vessel”labware vessels. This is because seeding a cell in a microfluidic cellculture device may often be done by microinjection via a hypodermicneedle device or connection, which is more difficult for the user andharder to standardize than traditional open-vessel cell culture wheremulti-channel pipetors or robotics are used. In addition, somemicrofluidic devices are composed of materials such aspolydimethylsiloxane (PDMS) because such materials are amenable toprototype microfabrication and are bio-inert. However, PDMSmicro-fabrication is difficult to mass produce. PDMS is also highlyadsorbent of lipophilic compounds, which presents problems for drugscreening applications where such lipophilic compounds are common.Furthermore, PDMS breathes, which presents a design complication forapplications requiring atmospheric control. In addition, interfacing tosuch devices may be problematic, particularly for placing experimentaldrugs traditionally stored in robotic compatible microplate devices intomicrofluidic structures. As such, microfluidic devices may only beavailable for certain applications such as protein crystallization,protein analysis, and PCR, but are not suited for cell analysis.

“Tissue on a chip” or “organ on a chip” applications are generally notsuited for in vitro cell culture as described herein because suchapplications lack inherent atmospheric control, low test chamberthroughput, and require recirculation of media instead of removing wastemedia. In addition, the cell chamber in such applications isinaccessible to integrated microscopy. Moreover, fluid perfusion of allsample bioreactors must occur in parallel.

Microfluidic cell culture devices that use a single standard“microplate-like” device having microfluidic channels that areincorporated into the device in order to move fluids from individualwells to specially constructed microfluidic cell chambers also built onthe device and use a pneumatic manifold to hermetically seal to the topof the plate to apply pneumatic pressure are also not suited for invitro cell culture as described herein. This is because the designincludes a low throughput, as there are few cell culture chambers (e.g.,four chambers) per plate. In addition, a passive gravity flow method asimplemented in such a design is inherently transient and defined by theever-changing fluid height difference between source and waste wells. Assuch, a user has no control of flow initiation or flow rate, whichlimits a duration of experimentation, as the fluid volume of the sourcewells are quickly expended. This, in turn, drives the need to attachmany source wells to a given cell chamber, thereby expanding experimentduration but limiting available cell chamber number for a given labvessel footprint. Other techniques to increase flow duration timeinclude adding mechanical interventions, such as tilting the plate tomechanically manipulate fluid height differences between source and sinkwells and thereby increase flow duration time, resulting in a reversalof the flow direction.

Devices that incorporate specially designed cell culture vessels matedto a fluid transfer base plate that is controlled by a computer are alsonot suited for in vitro cell culture as described herein because suchdevice's use non-traditional cell culture labware. Similarly, devicesthat are a hybrid, between traditional microplate culture, plates andmicrofluidic cell culture, plates, use a pneumatic pressure driven lidthat mates with, a traditional 24-well microplate, and moves fluid isfrom different wells of the microplate through integrated capillarytubes via a system of integrated valving and precisely timed airpressure and vacuum application are also not suited for in vitro cellculture as described herein. This is because such devices suffer fromlow throughput, use of only six measurement wells, requires perfusion ofall wells simultaneously, and the valving components utilize a flexiblePDMS layer to open and close the micro-channels, which, as previouslydescribed herein, is absorbent of lipophilic compounds and is permeableto gases, which are not controlled.

Referring now to the drawings, FIGS. 1A and 1B depict schematic views ofan illustrative apparatus, generally designated 100, for providingintegrated perfusion and atmospheric control of microplate labware. Morespecifically, FIG. 1A depicts the various components of the apparatus100 in an exploded schematic view and FIG. 1B depicts the variouscomponents of the apparatus 100 when assembled, as described in greaterdetail herein. In some embodiments, the apparatus 100 is housed within atemperature controlled environment 106, such as an incubator or thelike.

As particularly shown in FIG. 1B, the apparatus 100 may be positionedsuch that a bottom portion 102 thereof can be imaged via a microscopeobjective 154 of the microscope 150 and a corresponding top portion 104thereof is adjacent to a lamp housing 152 of the microscope 150. Assuch, the materials used for constructing various components of theapparatus 100 are sufficiently transparent so to allow light from thelamp housing 152 to illuminate the cells within the various wells 132.Accordingly, the apparatus 100 may be compatible with phase contrast orother transmissive light microscopy techniques. In some embodiments, theapparatus 100 may also be compatible with epi-fluorescence imagingdevices with excitation light entering the microscope objective from thebottom portion 102 of the apparatus 100.

Still referring to FIGS. 1A and 1B, the apparatus 100 generally includesa pneumatic lid 110 that is placed on various types of microplatelabware, including, but net limited to, a deep well source microplate120 and a cell assay microplate 130. The pneumatic lid 110 may be placedon an opening of the various microplate labware so as to seal themicroplate labware from an outside environment and/or to secure thepneumatic lid 110 to the microplate labware, as described in greaterdetail herein.

The pneumatic lid 110 may be, for example, a specially designed,sterile, pneumatic lid consumable. The pneumatic lid 110 includes a body111 having one or more pneumatic control fittings 108, one or more firstextension pieces 116, one or more second extension pieces 118, and oneor more microfluidic channels 114 fluidly coupling the one or more firstextension pieces 116 with the one or more second extension pieces 118.The one or more microfluidic channels 114 may generally provideperfusion capabilities between the various microplate labware via theone or more first extension pieces 116 and/or the one or more secondextension pieces 118. For example, when the apparatus 100 is assembledas shown in FIG. 1B, the first extension pieces 116 may extend into thedeep well source microplate 120 and the one or more second extensionpieces 118 may extend into the cell assay microplate 130. As such, inorder to increase the capacity of the apparatus 100 and yet not limitthe number of cell culture wells per device, the pneumatic lid 110 mayprovide a bridge between two or more microplates, such as the deep wellsource microplate 120 and the cell assay microplate 130, as depicted inFIG. 1B.

The pneumatic lid 110 may be constructed of any material, and isotherwise not limited by this disclosure. In some embodiments, thepneumatic lid 110 may be constructed of a plurality of layers ofmaterial. For example, the pneumatic lid may have a top layer 112 over amiddle layer comprising the microfluidic channels 114. In someembodiments, the pneumatic lid 110 may be constructed of materials thatdo not incorporate PDMS. In such embodiments, the materials may be morecompatible with the transfer of lipophilic drugs relative to materialsthat do incorporate PDMS. In addition, unlike PDMS, the materials usedin construction of the pneumatic lid 110 are not permeable to gases atleast to the extent that a gas composition of supplied air remainssubstantially unaltered. In some embodiments, the pneumatic lid 110 or aportion thereof (e.g., a portion of the body 111) may be constructed ofa thermoplastic elastomer (such as styrene ethylene butylene styrene(SEBS)) to make a reversible, yet gas impermeable, bond with themicroplates. SEBS is a thermoplastic elastomer (TPE) comprised of amixture of hard polymer such as polystyrene with ethylene-butylenechains. The ethylene-butylene chains give the material its flexibilityand the percentage composition of the hard polymer composition can becustomized depending on the desired characteristics required. The morepolystyrene used in the mix, the harder the material and the morechemically inert. The less polystyrene used in the mix, the softer thematerial and the less chemically inert. The advantages of SEBS overother compounds such as PDMS is that SEBS is less absorbent tolipophilic compounds and can easily and reversibly be bonded to glass,polystyrene, or itself without using solvents. SEBS compounds may alsobe less gag permeable than other compounds such as PDMS, which may bepreferable when building an environmentally closed system as describedherein. As such, SEBS may be more desirable than other compounds in someembodiments. Depending on the polystyrene composition, the material canbe injection molded or hot embossed.

The closed system as described herein may allow for atmospheric controland may eliminate various long term evaporation, and specificallynon-uniform evaporation, between the center wells and the edge wells ofa particular microplate. This type of evaporation may be common in othermicroplate lid designs which non-uniformly exchange air with anincubator environment. This air exchange and subsequent non-uniformevaporation can cause temperature variations as well as osmolaritychanges producing edge effects predominant in other microplate liddesigns.

In some embodiments, the pneumatic lid 110 may be devoid of mechanicalmeans, such as damps, pins, screws, or the like, for securing thepneumatic lid 110 to the microplate labware. Rather, the pneumatic lid110 may be secured to the microplate labware via any othernon-mechanical means, such as, for example, via vacuum pressure or viause of certain materials described herein. Use of non-mechanical meansto secure the pneumatic lid 110 to the microplate labware may beadvantageous over use of mechanical means because some mechanical meansare not reversible. As such, a user would not be able to remove the lidand reattach it to the same or other microplate labware.

Referring now to FIGS. 1C and 1D, in some embodiments, the pneumatic lid110 may have a plurality of portions 111 to facilitate coupling to themicroplate labware by allowing each section to be individually coupledto a corresponding microplate labware and then bridged together. Forexample, the pneumatic lid 110 may include a first portion 111 a thatcouples to the deep well source microplate 120, a second portion 111 cthat couples to the cell assay microplate 130 and is separate from thefirst portion 111 a, and a bridge portion 111 b that couples between thefirst portion 111 a and the second portion 111 c to fluidly connect thefirst portion 111 a to the second portion 111 c. As such, the pneumaticlid 110 may allow for a user to individually couple each of the firstportion 111 a and the second portion 111 c to their respectivemicroplate labware without hindering coupling of the other. Then, oncethe first portion 111 a and the second portion 111 c are coupled, thebridge portion 111 b is placed between the first portion 111 a and thesecond portion 111 c, as particularly shown in FIG. 1D. Accordingly, thebridge portion 111 b comprises bridge microfluidic channels 114 b thatalign with and fluidly couple to first microfluidic channels 114 a inthe first portion 111 a and second microfluidic channels 114 c in thesecond portion 111 c so that fluid flow is enabled through the firstmicrofluidic channels 114 a and the second microfluidic channels 114 cin a manner as described herein.

Referring again to FIGS. 1A and 1B, the microplate labware is generallystandard microplate labware as commonly understood, and is used as acell culturing device. Use of such microplate labware provides a largedegree of familiarity and experimental flexibility with respect toexisting cell culture work flow and methods. Illustrative examples ofmicroplate labware that may be used include, but are not limited to, thedeep well source microplate 120 and the cell assay microplate 130. Thedeep well source microplate 120 and the cell assay microplate 130 maybe, for example, microplates having a similar format and/orconfiguration. The deep well source microplate 120 may generally includeone or more wells 122 that are configured to contain a reagent. In someembodiments, the deep well source microplate 120 may be particularlyconfigured to increase reagent capacity and total perfusion time for agiven flow rate. In some embodiments, the deep well source microplatemay have a height of about 1 centimeter (cm) or greater. In variousembodiments, the cell assay microplate 130 may contain various cellsthat are to be studied. The cell assay microplate 130 may be, forexample, a microplate that also contains one or more wells 132. In aparticular embodiment, the cell assay microplate 130 may be a standard96-well microplate. However, other types of microplates should generallybe understood to be useful in this context and the type of the variousmicroplates is not limited to the present disclosure. However, for thesake of illustration, FIGS. 1A and 1B each depict a 96 well formatsource and destination plate in a cross-sectional view.

Fluid may move from the one or more wells 122 of the deep well sourcemicroplate 120 into the one or more wells 132 of the cell assaymicroplate 130 via the one or more first extension pieces 116, themicrofluidic channels 114 incorporated within the pneumatic lid 110,and/or the one or more second extension pieces 118. More specifically,the microfluidic channels 114 may utilize pneumatic pressure or a vacuumto effect fluid movement between microplates. The pneumatic pressure orthe vacuum may be introduced through the one or more pneumatic controlfittings 108.

As previously described herein, FIG. 1B depicts the pneumatic lid 110when engaged with the individual microplates (e.g., the deep well sourcemicroplate 120 and the cell assay microplate 130). When the pneumaticlid 110 is engaged with the microplates, an airtight seal 140 is formedbetween the pneumatic lid 110 and the microplates.

Such a closed (e.g., sealed) system as described herein may be necessaryto initiate pressure differentials (positive or negative) so as to movefluids between various components, control the rate of fluid flow,and/or control a duration of fluid flow. For example, the closed systemmay allow for fluid flow between the one or more wells 122 of the deepwell source microplate 120, the one or more wells 132 of the cell assaymicroplate 130, and/or the microfluidic channels 114 via the one or morefirst extension pieces 116 and/or the one or more second extensionpieces 118. In addition, sealing of the pneumatic lid 110 to themicroplates may be necessary in order to maintain the gas composition ofthe liquid reagents used in the apparatus 100, as described herein.

Referring also to FIG. 2, the pneumatic pressure or vacuum that isgenerated to form the airtight seal 140 may be controlled by a pneumaticcontroller 220 fluidly coupled to the one or more pneumatic controlfittings 108. As shown in FIG. 2, the pneumatic controller may generallybe a portion of a computing device that is configured to control thepneumatic-pressure and/or vacuum used to move fluids betweenmicroplates. The pneumatic controller 220 is configured such that eachsource well can be activated for flow independently, arbitrary grouped,or all activated simultaneously depending on the application. Thepneumatic controller 220 may also be configured to control a gascomposition of air that is supplied to the one or more pneumatic controlfittings 108. For example, the pneumatic controller 220 may control theoxygen partial pressure of the air supplied to the one or more pneumaticcontrol fittings 108. In some embodiments, the pneumatic controller 220may allow the pneumatic lid 110 to form the airtight seal 140 with themicroplates such that the fluid within the microplates equilibrates tothe constituency of the gas supplied. This provides a means ofcontrolling the dissolved oxygen (Or any other dissolved gas) content ofthe liquid reagents within the apparatus 100.

The various other hardware components, depicted in FIG. 2 may beparticularly configured to carry out various tasks for controlling theenvironment of the microplates once the pneumatic lid 110 has beenplaced thereover. A local interface 200 (such as a bus) may interconnectthe various components. A processing device 202, such as a computerprocessing unit (CPU) may be the central processing unit of thecomputing device, performing calculations and logic operations requiredto execute a program. The processing device 202, alone or in conjunctionwith one or more of the other elements disclosed in FIG. 2, is anillustrative processing device, computing device, processor, orcombination thereof, as such terms are used within this disclosure.Memory, such as read only memory (ROM) 206 and random access memory(RAM) 204, may constitute illustrative memory devices (i.e.,non-transitory processor-readable storage media). Such memory 204, 206may include one or more programming instructions thereon that, whenexecuted by the processing device 202, cause the processing device 202to complete various processes, such as the processes described herein.Optionally, the program instructions may be stored on a tangible,computer-readable medium such as a compact disc, a digital disk, flashmemory, a memory card, a USB drive, an optical disc storage medium, suchas a Bluray™ disc, and/or other non-transitory processor-readablestorage media.

A data storage device 208, which may generally be a storage medium thatis separate from the RAM 204 and the ROM 206, may contain a repositoryfor storing data such as pressure data or the like. The data storagedevice 208 may be any physical storage medium, including, but notlimited to, a hard disk drive (HDD), memory, removable storage, and/orthe like. While the data storage device 208 is depicted as a localdevice, it should be understood that the data storage device 208 may bea remote storage device, such as, for example, a server computingdevice, cloud based storage, and/or the like.

A user interface 212 may permit information from the local interface 200to be displayed on a display 214 portion of the computing device inaudio, visual, graphic, or alphanumeric format. Moreover, the userinterface 212 may also include one or more input devices 216 that allowfor transmission to and receipt of data from input devices such as akeyboard, a mouse, a joystick, a touch screen, a remote control, apointing device, a video input device, an audio input device, a hapticfeedback device, and/or the like. Such a user interface 212 may be used,for example, to allow a user to interact with the apparatus 100 toadjust a pressure or the like. For example, a user may interact with theapparatus 100 to provide experimental parameters to ensure that anappropriate environment is created on the microplates.

A system interface 218 may generally provide the computing device withan ability to interface with the pneumatic controller 220 and/or one ormore external components. Communication with the pneumatic controller220 and/or external components may occur using various communicationports (not shown). An illustrative communication port may be attached toa communications network, such as the Internet, an intranet, a localnetwork, a direct connection, and/or the like.

Controlled movement of fluid using pressure (or vacuum) through theapparatus 100 may be accomplished in one or more different manners. Forexample, in some embodiments, the apparatus 100 may include individualcontrol lines to each source well whereby the fluid flow can beactivated by the pneumatic controller to turn on/off the line pressureto each well. This approach may be advantageous because it allows for apneumatic lid 110 that is lacking any valves, which reduces thecomplexity of the design of the pneumatic lid 110. Rather, the valvesare located within the pneumatic controller 220. Such a design featuremay require pressurization and de-pressurization of the entire pneumaticcontrol line and head space above each reservoir well upon eachactivation.

In some embodiments, the apparatus 100 may incorporate a normally closedintegrated valve design, such as, for example, a Quake valve. Such anintegrated valve design may rely on a microfluidic channel configurationthat is partially comprised of a softer material which can be pushed(pressure) or pulled (vacuum) against a mating valve seat typically of aless flexible material to close or open and thereby turn off or turn onfluid flow in the microfluidic channel. Such valves can be configured asnormally open (open unless activated) or normally closed (closed unlessactivated) depending on the configuration. In some embodiments, theapparatus 100 may incorporate a normally closed valve such that fluidflow does not occur unless actuated by the pneumatic controller 220. Insuch embodiments, the normally closed valve may incorporate anelastomeric material that is amenable to microfabrication and is alsobio-inert.

In some embodiments, SEBS may be used as an elastomeric deflectivematerial for an integrated normally closed valve design. For example, adesign that incorporates integrated valves fluidly coupled to channelsthat are embossed or injection molded SEBS material and mated to a lessflexible, material like polystyrene (PS) or cyclin olefin polymer (COP)in completing the channel. In other embodiments, various channelfeatures may be constructed of PS or COC, with valve actuationcomponents constructed of SEBS.

In various embodiments, the polystyrene composition of the SEBS used inthe various components of the apparatus 100 may be varied and optimizedto address the various requirements. For example, requirements offorming a reversible bond not requiring solvent, a layer that maintainsseal in the presence of the pressures required for perfusion function, abonding layer that is configured for mating to standard microplatelabware while maintaining various mechanical tolerances, and a bondinglayer which is not permeable to gases for the purpose of maintaining gascomposition of the media may be addressed.

FIG. 3A depicts a schematic view of an illustrative pneumatic lid thatincorporates solid polymer plugs at a destination plate interface. Inthe embodiment depicted in FIG. 3A, a pneumatic lid 310 may include abody 311 having four polymer layers. A top layer 312 of the pneumaticlid 310 is comprised of a harder polymer (polystyrene, cyclic olefinpolymer, and/or the like). The top layer 312 may contain one or morepneumatic control fittings 308 that are fluidly coupled to a pneumaticcontroller (not shown), such as the pneumatic controller 220 (FIG. 2)described hereinabove. In some embodiments, the top layer 312 maycontain one or more pneumatic air channels that run spatially across thepneumatic lid 310 for individual valve control and/or wellpressurization. The pneumatic lid 310 may further include a second layer313 underneath the top layer 312. The second layer 313 may be comprisedof a thermoplastic elastomer (TPE), such as SEBS or the like. The secondlayer 313 may be utilized as a deflection layer for an integrated valvedesign. In some embodiments, the second layer 311 may not containmicrofluidic channels (featureless). Rather, the second layer 313 may bemated to a third layer 314 constructed of a polymer and containingembossed or injection molded microfluidic channels therein. In such anarrangement, the second layer 313 may be sandwiched between the toplayer 312 and the third layer 314. The microfluidic channels locatedwithin the third layer 314 may have lateral dimensions of about 30microns to several hundred microns. One or more extension piecesextending from the third layer 314 may provide an interface to theindividual microplates. For example, in some embodiments, an interfacebetween the microfluidic channels in the third layer 314 and a sourceplate may include one or more capillary tubes 316. In some embodiments,the one or more capillary tubes 316 may be injection molded as a portionof the third layer 314. In other embodiments, the one or more capillarytubes 316 may be inserted separately into the third layer 314.

FIGS. 3A and 3B depict another embodiment where the apparatus, generallydesignated 300, incorporates a pneumatic lid 310. Except as specificallydescribed herein, the various remaining components shown in FIGS. 3A and3B may be constructed and configured similar to the like-numberedcomponents in FIGS. 1A and 1B. For example, the top layer 312 depictedin FIG. 3A may be constructed and configured in a manner similar to thetop layer 112 described with respect to FIG. 1A.

As shown in FIGS. 3A and 3B, a destination cell plate 330 may have oneor more wells 332 that have extension pieces from the third layer 314inserted therein. Such extension pieces may be, for example, solidpolymer plugs 318 that contain one or more bores 319 therein. The one ormore bores 319 may function as inlet and/or outlet fluid paths into arespective well 332 of the destination cell plate 330. Use of such plugs318 may provide an advantage over other apparatuses as fluid levels atthe bottom of each well 332 in the destination cell plate 330 can beconfined. In addition, use of such plugs 318 may eliminate a meniscus inthe fluid contained in each well 332. Elimination of a meniscus mayallow for more accurate imaging of the contents of each well 332, as ameniscus may cause artifacts that are detrimental to microscopicimaging. In some embodiments, as particularly shown in FIG. 3B, a bottomportion B of each plug 318 may be particularly shaped and/or sized. Sucha particular shape and/or size of the bottom portion B may be generallyfor the purposes of priming and bubble removal. In addition, the shapeand/or size of the bottom portion B may aid in providing an ease ofdesigning and manufacturing of the plug 318.

In some embodiments, the pneumatic lid 310 may also incorporate a fourthlayer 315 underneath the third layer 314, such that the third layer 314is positioned between the second layer 313 and the fourth layer 315. Thefourth layer may be comprised of a thermoplastic elastomer, and maygenerally be used to provide a sealing surface for attachment to theindividual microplates as described herein.

FIGS. 4A and 4B depict another embodiment where the apparatus, generallydesignated 400, incorporates a pneumatic lid 410 having polymer tubes417 located at an interface with a destination microplate 430. Except asspecifically described herein, the various remaining components shown inFIGS. 4A and 4B may be constructed and configured similar to thelike-numbered components in FIGS. 1A, 1B, 3A, and 3B. For example, thebody 411 depicted in FIG. 4A may be constructed and configured in amanner similar to the body 111 described with respect to FIGS. 1A and 1Band the body 311 described with respect to FIG. 3A. In another example,the first layer 412 depicted in FIG. 4A may be constructed andconfigured in a manner similar to the top layer 312 described withrespect to FIG. 3A.

In the embodiment depicted in FIGS. 4A and 4B, the fluid interface tothe destination microplate 430 may be provided via the polymer tubes417, which are fluidly coupled to microfluidic channels located within athird layer 414. As particularly shown in FIG. 4B, a first tube 417 amay provide a fluid inlet into each well 432 of the destinationmicroplate 430 and a second tube 417 b may provide a fluid outlet fromeach well 432 of the destination microplate 430. In some embodiments,the first tube 417 a may extend a distance into a respective well 432that is different from a second distance at which the second tube 417 bextends. As such, the various tubes 417 may be located at variousheights within each well 432. In some embodiments, each of the varioustubes 417 may be controlled by separate valving or common valving, andeach of the various tubes 417 may be actuated by pressure or vacuum, asdescribed in greater detail herein.

Use of microfluidic channels in the pneumatic lid as described hereinmay provide for design flexibility with respect to interfacingparticular source wells with particular destination wells. For exampleas shown in FIGS. 5A-5C, several well-to-well mapping possibilities mayexist.

FIG. 5A depicts a direct one to one mapping. As such, each of the sourcewells 522 a, 522 b has a corresponding valve 560 a, 560 b fluidlycoupled thereto. Each valve 560 a, 560 b is fluidly coupled to acorresponding destination well 532 a, 532 b via a corresponding conduit570, 580. As such, fluid contained within a first source well 522 a isselectively controlled by a first valve 560 a to move fluid through afirst conduit 570 into a corresponding first destination well 532 a.Similarly, fluid contained within a second source well 522 b isselectively controlled by a second valve 560 b to move fluid through asecond conduit 580 into a corresponding second destination well 532 b.

In other embodiments, as shown in FIG. 5B, two source wells may beinterfaced to either of two destination wells. More specifically, eachof the source wells 522 a, 522 b has a corresponding valve 560 a, 560 bfluidly coupled thereto. Each valve 560 a, 560 b is fluidly coupled toall of a plurality of destination wells 532 a, 532 b via a communalconduit 590. As such, fluid contained within a first source well 522 ais selectively controlled by a first valve 560 a to move fluid throughthe communal conduit 590 into a first destination well 532 a and/or asecond destination well 532 b. Similarly, fluid contained within asecond source well 522 b is selectively controlled by a second valve 560b to move fluid through the communal conduit 590 into the firstdestination well 532 a and/or the second destination well 532 b. In thisembodiment, a particular destination well can receive more than onereagent, which may offer experimental flexibility as well as redundancy(duplicates).

FIG. 5C depicts an illustrative example of quadruplicate well mapping.FIG. 5C depicts four source wells 522 a, 522 b, 522 c, 522 d fluidlycoupled to a corresponding valve 560 a, 560 b, 560 c, 560 d, each ofwhich is fluidly coupled to all of a plurality of destination wells 532a, 532 b, 532 c, 532 d via a communal conduit 590. The various wells andvalves operate in a manner similar to those described with respect toFIG. 5B. In this embodiment, a particular destination well, can receivemore than one reagent, which may offer experimental flexibility as wellas redundancy (duplicates). Other configurations not specificallydescribed herein may also be possible without departing from the scopeof the present disclosure. Also, as previously mentioned herein, activevalves may not be used. Rather, the actuation of fluid flow may beaccomplished via a separate pressurization of an isolated well pneumaticline from the pneumatic controller. In this instance however, it may bedesirable to incorporate a simple passive check valve to preventback-flow from one source well into another source well in the contextof multiple well mapping scenarios as described in FIGS. 5A-5C.

In various embodiments, it may be necessary to ensure that cellscontained within destination wells do not experience significantdeviations from atmospheric pressure in the process of moving fluidswithin the apparatus. When it is settled and pressurized as describedherein. As such, it may be necessary to introduce pressure differentials(positive or negative) in the source wells and the destination wellswithout introducing significant (<0.1 atm) deviations from absoluteatmospheric pressures in the cell microplate. This may be incorporatedin the design of the apparatus and the various components thereof, andin some embodiments may be based on fluid channel geometry, valving,and/or various pressures used at the pneumatic controller.

In various embodiments, waste removal may be necessary to eliminate anartificial condition of extraneous waste build-up at the cell layer, andmay be in concordance with the in vivo condition of a steady-stateconcentration of nutrients and waste as previously described herein. Insome embodiments, the volume of material in each well remains constantand the fluid volume removed is equivalent to that which is added. FIGS.6A and 6B depict illustrative apparatus configurations that are suitedfor waste removal. For example, as shown in FIG. 6A, waste may becarried out to a waste well 622 b via one or more conduits 670, 680. Inanother example, as shown in FIG. 6B, a source well 622 a may be fluidlycoupled to a first valve 650 a, which controls fluid flow via a firstconduit 670 to a first destination well 632 a and/or a seconddestination well 632 b. The waste well 622 b may be fluidly coupled to asecond valve 650 a, which controls fluid flow via a second conduit 680to the first destination well 632 a and/or the second destination well632 b. In such an embodiment, the source well 622 a may be left empty atthe beginning of an experiment and subsequently used as a wastecollection vessel. This may be useful in situations where it may bedesirable to keep the perfusion waste from various wells for subsequent(e.g., biochemical) analysis. In some embodiments, the timing andvolumes of adding to the wells and the related timing and volumes ofwaste extraction may be varied to take advantage of other factors suchas convection, diffusional mixing, and/or the like.

It should be appreciated that methods of assembling the variousapparatuses described hereinabove may include various steps such as, butnot limited to, providing the microplates and placing the pneumatic lidover the microplates (including placing the portions of the pneumaticlid and the corresponding bridge portion). It should further beappreciated that methods may include inserting extension pieces into thewells of the corresponding microplates to fluidly couple the microplatesto one another. The lid may be coupled via a non-mechanical device, asdescribed herein. In addition, a differential pressure may be activatedto control fluid flow.

It should now be understood that the systems, apparatuses, and methodsdescribed herein incorporate a pneumatic lid with traditional microplatecomponents for the purposes of simulating in vivo conditions forcellular cultures. The systems, apparatuses, and methods describedherein use traditional microplate technology, such that it can be usedwith current work flow and readout technologies. Moreover, use oftraditional microplate technology eliminates difficulty with seedingcells in microfluidic channel devices. The systems, apparatuses, andmethods described herein provide a capacity for a greater experimentalthroughput relative to other technologies, an ability to controldissolved gas composition of the cell culture media (e.g., oxygencomposition), an ability to form a solvent-free, non-breathing bond,which results in a closed design that eliminates evaporation andresulting edge effects associated with other microplate cultures, adevice that is capable of integrated perfusion while simultaneouslybeing accessible to microscopic imaging, allows for independent wellcontrol (ability to perfuse individual wells, groups of wells, or allwells in unison), ability to use active flow whereby flow rate and flowduration can be controlled by the user, ability to provide an enhancedexperimental duration for a given flow rate via a deep well sourcemicroplate, and/or an apparatus that eliminates or minimizes the use ofPDMS material, instead incorporating materials such as SEBS to provideenhanced chemical resistance and/or gas permeability.

EXAMPLES

Illustrative examples of potential uses of the systems, apparatuses, andmethods described above are provided below. Such examples are merelyillustrative in nature and are not intended to limit the scope of thepresent disclosure. In addition, the list of illustrative examplesprovided below is not exhaustive and may include other examples withoutdeparting from the scope of the present disclosure.

Such a device or technique could be used to feed cells in an automatedmanner, using more physiological concentrations of nutrients whilesimultaneously achieving a more biologically relevant, steady-stateconcentration of nutrients and waste products. As described, this methodwould benefit almost all known in vitro cell models and would beapplicable in broad fields of life science research including drugdiscovery and safety testing.

Such a device or technique could be used to discover, design, andvalidate media formulations which are more consistent with physiologicalconditions.

Such a device or technique could be used to more easily investigate andoptimize the timing and composition of media constituents in studyingstem cell differentiation.

Such a device or technique could be used to more easily investigate theeffects of gas composition, e.g., oxygen concentration on various invitro cell models such as neurons or hepatocytes.

Such a device or technique could be used to add a cell modulatingreagent while simultaneously imaging the cells (e.g., acute drugexposure studies)

Such a device or technique could be used to add drugs or other cellmodulators via the media in a manner which is more physiological indelivery and less perturbing to the cultures than removing the platefrom the incubator.

Such a device or technique could be used to change media constituentsfrom reagent A to reagent B, and/or remove a drug or media constituent(wash-out) from the culture.

Such a device or technique could be used to mimic or model the drugmetabolism and pharmokinetic concentration profile of a drug, agent ormetabolite by automatically changing the concentration of the agent overtime.

Such a device or technique could be used to sample waste products fromthe cells for further analysis.

Such a device or technique which would allow for perfusion of one well,arbitrary groups of wells, or all the wells of a standard microplate.

Such a device or technique where the pneumatic lid assembly is packagedas a sterile consumable and applicable to sterile cell culturetechniques.

Item List

Item 1. A pneumatic lid comprising:

a body comprising one or more microfluidic channels, wherein at least aportion of the body is constructed of styrene ethylene butylene styrene(SEBS);

one or more first extension pieces fluidly coupled to the one or moremicrofluidic channels and extending from the body; and

one or more second extension pieces fluidly coupled to the one or moremicrofluidic channels and extending from the body.

Item 2. A pneumatic lid comprising:

a first portion comprising one or more first microfluidic channels thatare configured to be fluidly coupled to one or more first wells;

a second portion comprising one or more second microfluidic channelsthat are configured to be fluidly coupled to one or more second wellsthat are separate from the one or more first wells; and

a removable bridge portion extending between the first portion and thesecond portion, wherein the removable bridge portion, when coupled tothe first portion and the second portion, fluidly couples the one ormore first microfluidic channels to the one or more second microfluidicchannels,wherein the first portion and the second portion, when coupled to theone or more first wells and the one or more second wells, respectivelyprovide an airtight seal over the one or more first wells and the one ormore second wells.

Item 3. The pneumatic lid of item 2, wherein the pneumatic lid isconstructed of styrene ethylene butylene styrene (SEBS).

Item 4. The pneumatic lid of item 1 or 2, wherein the first portion andthe second portion, when coupled to the one or more first wells and theone or more second wells, respectively provide the airtight seal overthe one or more first wells and the one or more second wells via anon-mechanical device.

Item 5. The pneumatic lid of any one of items 2 to 4, further comprisingone or more valves that selectively control fluid flow within the one ormore microfluidic channels.

Item 6. The pneumatic lid of any one of items 2 to 5, wherein a fluidflow rate and a duration are controlled by activation of a differentialpressure.

Item 7. The pneumatic lid of any one of items 2 to 6, wherein theairtight seal is a reversible airtight seal.

Item 8. The pneumatic lid of any of items 2 to 7, further comprising oneor more pneumatic control fittings fluidly coupled to at least a portionof the pneumatic lid.

Item 9. An apparatus comprising:

a first microplate having a first open portion and defining one or morefirst wells therein;

a second microplate having a second open portion and defining one ormore second wells therein; and

a pneumatic lid constructed of styrene ethylene, butylene styrene(SEBS), the pneumatic lid extending over the first open portion and thesecond open portion and comprising one or more microfluidic channelsthat fluidly couple the one or more first wells to the one or moresecond wells, wherein the pneumatic lid provides an airtight seal overthe first microplate and the second microplate.

Item 10. An apparatus comprising:

a first microplate having a first open portion and defining one or morefirst wells therein;

a second microplate having a second open portion and defining one ormore second wells therein; and

a pneumatic lid extending over the first open portion and the secondopen portion, the pneumatic lid comprising one or more microfluidicchannels that fluidly couple the one or more first wells to the one ormore second wells, wherein the pneumatic lid provides an airtight sealover the first microplate and the second microplate.

Item 11. An apparatus comprising:

a first microplate having a first open portion and defining one or morefirst wells therein;

a second microplate having a second open portion and defining one ormore second wells therein; and

a pneumatic lid comprising:

a first portion extending over the first open portion, the first portioncomprising one or more first microfluidic channels that are fluidlycoupled to the one or more first wells,

a second portion extending over the second open portion, the secondportion comprising one or more second microfluidic channels that arefluidly coupled to the one or more second wells, and

a removable bridge portion extending between the first portion and thesecond portion, wherein the removable bridge portion, when coupled tothe first portion and the second portion, fluidly couples the one ormore first microfluidic channels to the one or more second microfluidicchannels,wherein the pneumatic lid provides an airtight seal over the firstmicroplate and the second microplate.

Item 12. The apparatus of item 10 or item 11, wherein the pneumatic lidis constructed of a thermoplastic elastomer that forms a reversible andgas impermeable bond with the first microplate and the secondmicroplate.

Item 13. The apparatus of item 12, wherein the thermoplastic elastomeris styrene ethylene butylene styrene (SEBS).

Item 14. The apparatus of any one of items 9-13, wherein the pneumaticlid further comprises:

one or more first extension pieces extending into the one or more firstwells of the first microplate; and

one or more second extension pieces extending into the one or moresecond wells of the second microplate,

wherein the one or more microfluidic channels fluidly couple the one ormore first extension pieces to the one or more second extension pieces.

Item 15. The apparatus of any one of items 9-13, wherein the pneumaticlid further comprises:

one or more extension pieces extending into the one or more first wellsof the first microplate; and

one or more polymer plugs extending into the one or more second wells ofthe second microplate, each one of the one or more polymer plugscomprising one or more bores therein,

wherein the one or more microfluidic channels fluidly couple the one ormore extension pieces to the one or more bores.

Item 16. The apparatus of any one of items 9, 10, or 12 to 15 whereinthe pneumatic lid comprises:

a first portion extending over the first open portion, the first portioncomprising one or more first microfluidic channels that are fluidlycoupled to the one or more first wells,

a second portion extending over the second open portion, the secondportion comprising one or more second microfluidic channels that arefluidly coupled to the one or more second wells, and

a removable bridge portion extending between the first portion and thesecond portion, wherein the removable bridge portion, when coupled tothe first portion and the second portion, fluidly couples the one ormore first microfluidic channels to the one or more second microfluidicchannels.

Item 17. The apparatus of any one of items 9-16, wherein the firstmicroplate does not contact the second microplate.

Item 18. The apparatus of any one of items 9-17, wherein the pneumaticlid is coupled to the first microplate and the second microplate via anon-mechanical device.

Item 19. The apparatus of any one of items 9-18, further comprising oneor more valves that selectively control fluid flow within the one ormore microfluidic channels.

Item 20. The apparatus of any one of items 9-19, wherein a fluid flowrate and a duration are controlled by activation of a differentialpressure.

Item 21. The apparatus of any one of items 9-20, wherein the airtightseal is a reversible airtight seal.

Item 22. The apparatus of any one of items 9-21, further comprising oneor more pneumatic control fitting fluidly coupled to at least a portionof the pneumatic lid.

Item 23. The apparatus of item any one of items 9-22, wherein:

the pneumatic lid further comprises a thermoplastic elastomer layer; and

the airtight seal is created via the thermoplastic elastomer layer.

Item 24. The apparatus of any one of items 9-23, wherein at least one ofthe first microplate and the second microplate comprises a deep wellplate having a height greater than 1 cm.

Item 25. A method of constructing an apparatus for transferring fluid,the method comprising:

providing a first microplate having a first open portion and definingone or more first wells therein;

providing a second microplate having a second open portion and definingone or more second wells therein; and

placing a pneumatic lid constructed of styrene ethylene butylene styrene(SEBS) over the first open portion and the second open portion such thatone or more microfluidic channels within the pneumatic lid are fluidlycoupled to the one or more first wells and the one or more second wells,wherein the pneumatic lid provides an airtight seal over the firstmicroplate and the second microplate.

Item 26. The method of item 25, wherein placing the pneumatic lidcomprises:

inserting one or more first extension pieces into the one or more firstwells of the first microplate; and

inserting one or more second extension pieces into the one or moresecond wells of the second microplate,

wherein the one or more microfluidic channels fluidly couple the one ormore first extension pieces to the one or more second extension pieces.

Item 27. A method of constructing an apparatus for transferring fluid,the method comprising:

providing a first microplate having a first open portion and definingone or more first wells therein;

providing a second microplate having a second open portion and definingone or more second wells therein;

placing a first portion of a pneumatic lid over the first open portionsuch that one or more first microfluidic channels within the firstportion are fluidly coupled to the one or more first wells;

placing a second portion of a pneumatic lid over the second open portionsuch that one or more second microfluidic channels within the secondportion are fluidly coupled to the one or more second wells; and

placing a removable bridge portion between the first portion and thesecond portion of the pneumatic lid to fluidly couple the one or morefirst microfluidic channels to the one or more second microfluidicchannels.

Item 28. The method of item 27, wherein:

placing the first portion of the pneumatic lid comprises inserting oneor more first extension pieces into the one or more first wells of thefirst microplate; and

placing the second portion of the pneumatic lid comprises inserting oneor more second extension pieces into the one or more second wells of thesecond microplate.

Item 29. The method of any one of items 25-28, wherein placing thesecond microplate comprises placing the second microplate at a distancefrom the first microplate such that the second microplate does notcontact the first microplate.

Item 30. The method of any one of items 25-29, wherein placing thepneumatic lid, or a portion thereof, comprises coupling the pneumaticlid to the first microplate and the second microplate via anon-mechanical device.

Item 31. The method of any one of items 25-30, further comprisingactivating a differential pressure to control fluid flow within the oneor more microfluidic channels.

Item 32. A system for transferring fluid, the system comprising:

a first microplate having a first open portion and defining one or morefirst wells therein;

a second microplate that is separate from the first microplate, thesecond microplate having a second open portion and defining one or moresecond wells therein;

a pneumatic lid constructed of styrene ethylene butylene styrene (SEBS)which forms a reversible and gas impermeable bond with the firstmicroplate and the second microplate, the pneumatic lid comprising:

a first portion extending over the first open portion, the first portioncomprising one or more first extension pieces extending into the one ormore first wells of the first microplate and one or more firstmicrofluidic channels that are fluidly coupled to the one or more firstwells via the one or more first extension pieces,a second portion extending over the second, open portion, the secondportion comprising one or more second extension pieces extending intothe one, or more second wells of the second microplate and one or moresecond microfluidic channels that are fluidly coupled to the one or moresecond wells via the one or more second microfluidic channels, anda removable bridge portion extending between the first portion and thesecond portion, wherein the removable bridge portion, when coupled tothe first portion and the second portion, fluidly couples the one ormore first microfluidic channels to the one or more second microfluidicchannels; andone or more valves fluidly coupled to the pneumatic lid, the one or morevalves configured to selectively control fluid flow within the one ormore first microfluidic channels and the one or more second microfluidicchannels,wherein the fluid is transferred between the first microplate and thesecond microplate via the pneumatic lid and the one or more valves.

Item 33. An apparatus for transferring fluid from a first standardmicroplate to a second standard microplate according to one or more ofthe embodiments described herein.

Item 34. A system for transferring fluid from a first standardmicroplate to a second standard microplate according to one or more ofthe embodiments described herein.

Item 35. A method for transferring fluid from a first standardmicroplate to a second standard microplate according to one or more ofthe embodiments described herein.

While particular embodiments have been illustrated and described herein,it should be understood that various other changes and modifications maybe made without departing from the spirit and scope of the claimedsubject matter. Moreover, although various aspects of the claimedsubject matter have been described herein, such aspects need not beutilized in combination. It is therefore intended that the appendedclaims cover all such changes and modifications that are within thescope of the claimed subject matter.

What is claimed is:
 1. A pneumatic lid comprising: A) a body comprisingone or more microfluidic channels, wherein at least a portion of thebody is constructed of styrene ethylene butylene styrene (SEBS); one ormore first extension pieces fluidly coupled to the one or moremicrofluidic channels and extending from the body; and one or moresecond extension pieces fluidly coupled to the one or more microfluidicchannels and extending from the body, wherein the one or more secondextension pieces are further fluidly coupled to the one more firstextension pieces via the one or more microfluidic channels; or (B) afirst portion comprising one or more first microfluidic channels thatare configured to be fluidly coupled to one or more first wells; asecond portion comprising one or more second microfluidic channels thatare configured to be fluidly coupled to one or more second wells thatare separate from the one or more first wells; and a removable bridgeportion extending between the first portion and the second portion,wherein the removable bridge portion, when coupled to the first portionand the second portion, fluidly couples the one or more firstmicrofluidic channels to the one or more second microfluidic channels,wherein the first portion and the second portion, when coupled to theone or more first wells and the one or more second wells, respectivelyprovide an airtight seal over the one or more first wells and the one ormore second wells.
 2. The pneumatic lid of claim 1, wherein thepneumatic lid comprises option (B), and wherein the first portion andthe second portion, when coupled to the one or more first wells and theone or more second wells, respectively provide the airtight seal overthe one or more first wells and the one or more second wells via anon-mechanical device.
 3. The pneumatic lid of claim 1, furthercomprising one or more valves that selectively control fluid flow withinthe one or more microfluidic channels.
 4. The pneumatic lid of claim 1,wherein a fluid flow rate and a duration are controlled by activation ofa differential pressure.
 5. The pneumatic lid of claim 1, wherein thepneumatic lid comprises option (B), and wherein the airtight seal is areversible airtight seal.
 6. The pneumatic lid of claim 1, furthercomprising one or more pneumatic control fittings fluidly coupled to atleast a portion of the pneumatic lid.
 7. An apparatus comprising: (A) afirst microplate having a first open portion and defining one or morefirst wells therein; a second microplate having a second open portionand defining one or more second wells therein; and a pneumatic lidconstructed of styrene ethylene butylene styrene (SEBS), the pneumaticlid positioned on top of the first microplate and the second microplatesuch that the pneumatic lid extends over the first open portion and thesecond open portion, the pneumatic lid comprising one or moremicrofluidic channels that fluidly couple the one or more first wells tothe one or more second wells when the pneumatic lid is positioned overthe first open portion and the second open portion, wherein thepneumatic lid provides an airtight seal over the first microplate andthe second microplate; or (B) a first microplate having a first openportion and defining one or more first wells therein; a secondmicroplate having a second open portion and defining one or more secondwells therein; and a pneumatic lid positioned on top of the firstmicroplate and the second microplate such that the pneumatic lid extendsover the first open portion and the second open portion, the pneumaticlid comprising one or more microfluidic channels that fluidly couple theone or more first wells to the one or more second wells when thepneumatic lid is positioned over the first open portion and the secondopen portion, wherein the pneumatic lid provides an airtight seal overthe first microplate and the second microplate; or (C) a firstmicroplate having a first open portion and defining one or more firstwells therein; a second microplate having a second open portion anddefining one or more second wells therein; and a pneumatic lidcomprising: a first portion extending over the first open portion, thefirst portion comprising one or more first microfluidic channels thatare fluidly coupled to the one or more first wells, a second portionextending over the second open portion, the second portion comprisingone or more second microfluidic channels that are fluidly coupled to theone or more second wells, and a removable bridge portion extendingbetween the first portion and the second portion, wherein the removablebridge portion, when coupled to the first portion and the secondportion, fluidly couples the one or more first microfluidic channels tothe one or more second microfluidic channels, wherein the pneumatic lidprovides an airtight seal over the first microplate and the secondmicroplate.
 8. The apparatus of claim 7, wherein the pneumatic lid isconstructed of a thermoplastic elastomer that forms a reversible and gasimpermeable bond with the first microplate and the second microplate. 9.The apparatus of claim 8, wherein the thermoplastic elastomer is styreneethylene butylene styrene (SEBS).
 10. The apparatus of claim 7, whereinthe pneumatic lid further comprises: one or more first extension piecesextending into the one or more first wells of the first microplate; andone or more second extension pieces extending into the one or moresecond wells of the second microplate, wherein the one or moremicrofluidic channels fluidly couple the one or more first extensionpieces to the one or more second extension pieces.
 11. The apparatus ofclaim 7, wherein the pneumatic lid further comprises: one or moreextension pieces extending into the one or more first wells of the firstmicroplate; and one or more polymer plugs extending into the one or moresecond wells of the second microplate, each one of the one or morepolymer plugs comprising one or more bores therein, wherein the one ormore microfluidic channels fluidly couple the one or more extensionpieces to the one or more bores.
 12. The apparatus of claim 7, whereinthe pneumatic lid comprises: a first portion extending over the firstopen portion, the first portion comprising one or more firstmicrofluidic channels that are fluidly coupled to the one or more firstwells, a second portion extending over the second open portion, thesecond portion comprising one or more second microfluidic channels thatare fluidly coupled to the one or more second wells, and a removablebridge portion extending between the first portion and the secondportion, wherein the removable bridge portion, when coupled to the firstportion and the second portion, fluidly couples the one or more firstmicrofluidic channels to the one or more second microfluidic channels.13. The apparatus of claim 7, wherein the first microplate does notcontact the second microplate.
 14. The apparatus of claim 7, wherein thepneumatic lid is coupled to the first microplate and the secondmicroplate via a non-mechanical device.
 15. The apparatus of claim 7,further comprising one or more valves that selectively control fluidflow within the one or more microfluidic channels.
 16. The apparatus ofclaim 7, wherein a fluid flow rate and a duration are controlled byactivation of a differential pressure.
 17. The apparatus of claim 7,wherein the airtight seal is a reversible airtight seal.
 18. Theapparatus of claim 7, further comprising one or more pneumatic controlfittings fluidly coupled to at least a portion of the pneumatic lid. 19.The apparatus of claim 7, wherein: the pneumatic lid further comprises athermoplastic elastomer layer; and the airtight seal is created via thethermoplastic elastomer layer.
 20. The apparatus of claim 7, wherein atleast one of the first microplate and the second microplate comprises adeep well plate having a height greater than 1 cm.
 21. A method ofconstructing an apparatus for transferring fluid, the method comprising:(A) providing a first microplate having a first open portion anddefining one or more first wells therein; providing a second microplatehaving a second open portion and defining one or more second wellstherein; and placing a pneumatic lid constructed of styrene ethylenebutylene styrene (SEBS) on top of the first microplate and the secondmicroplate such that the pneumatic lid extends over the first openportion and the second open portion such that one or more microfluidicchannels within the pneumatic lid are fluidly coupled to the one or morefirst wells and the one or more second wells when the pneumatic lid ispositioned over the first open portion and the second open portion,wherein the pneumatic lid provides an airtight seal over the firstmicroplate and the second microplate; or (B) providing a firstmicroplate having a first open portion and defining one or more firstwells therein; providing a second microplate having a second openportion and defining one or more second wells therein; placing a firstportion of a pneumatic lid over the first open portion such that one ormore first microfluidic channels within the first portion are fluidlycoupled to the one or more first wells; placing a second portion of apneumatic lid over the second open portion such that one or more secondmicrofluidic channels within the second portion are fluidly coupled tothe one or more second wells; and placing a removable bridge portionbetween the first portion and the second portion of the pneumatic lid tofluidly couple the one or more first microfluidic channels to the one ormore second microfluidic channels.
 22. The method of claim 21, whereinthe method comprises option (A), and wherein placing the pneumatic lidcomprises: inserting one or more first extension pieces into the one ormore first wells of the first microplate; and inserting one or moresecond extension pieces into the one or more second wells of the secondmicroplate, wherein the one or more microfluidic channels fluidly couplethe one or more first extension pieces to the one or more secondextension pieces.
 23. The method of claim 21, wherein the methodcomprises option (B), and wherein: placing the first portion of thepneumatic lid comprises inserting one or more first extension piecesinto the one or more first wells of the first microplate; and placingthe second portion of the pneumatic lid comprises inserting one or moresecond extension pieces into the one or more second wells of the secondmicroplate.
 24. The method of claim 21, wherein placing the secondmicroplate comprises placing the second microplate at a distance fromthe first microplate such that the second microplate does not contactthe first microplate.
 25. The method of claim 21, wherein placing thepneumatic lid, or a portion thereof, comprises coupling the pneumaticlid to the first microplate and the second microplate via anon-mechanical device.
 26. The method of claim 21, further comprisingactivating a differential pressure to control fluid flow within the oneor more microfluidic channels.
 27. A system for transferring fluid, thesystem comprising: a first microplate having a first open portion anddefining one or more first wells therein; a second microplate that isseparate from the first microplate, the second microplate having asecond open portion and defining one or more second wells therein; apneumatic lid constructed of styrene ethylene butylene styrene (SEBS)which forms a reversible and gas impermeable bond with the firstmicroplate and the second microplate, the pneumatic lid comprising: afirst portion extending over the first open portion, the first portioncomprising one or more first extension pieces extending into the one ormore first wells of the first microplate and one or more firstmicrofluidic channels that are fluidly coupled to the one or more firstwells via the one or more first extension pieces, a second portionextending over the second open portion, the second portion comprisingone or more second extension pieces extending into the one or moresecond wells of the second microplate and one or more secondmicrofluidic channels that are fluidly coupled to the one or more secondwells via the one or more second microfluidic channels, and a removablebridge portion extending between the first portion and the secondportion, wherein the removable bridge portion, when coupled to the firstportion and the second portion, fluidly couples the one or more firstmicrofluidic channels to the one or more second microfluidic channels;and one or more valves fluidly coupled to the pneumatic lid, the one ormore valves configured to selectively control fluid flow within the oneor more first microfluidic channels and the one or more secondmicrofluidic channels, wherein the fluid is transferred between thefirst microplate and the second microplate via the pneumatic lid and theone or more valves.